Historically, providing mama cell cultures with ideal growing conditions has been difficult. These delicate cells need low turbulence and high mass transfer of nutrients for proliferation and differentiation Bioreactors of the past have usually sacrificed one parameter for the other. Traditionally, stirring bars, bubble sparging and other mechanical means have promoted nutrient dispersion but also created significant shear stress and disruptive forces. Continuous shear stress causes the many cells to exit the proliferation or differentiation phases and activate survival mechanisms which may involve substantial cell death.
Another problem with current bioreactors is that there is no provision to separate the cells from the valuable bioproducts that they produce. Separating cells and cell debris from the secreted product is very labor intensive. Downstream processing costs account for roughly two-thirds of the manufacturing costs for secreted products.
A bioreactor to counteract a significant amount of shears stress while allowing high nutrient transfer and removal of waste and/or valuable bio-products, e.g., protein, hormones, growth factors, and monoclonal antibodies without disturbing the cell culture would be very desirable.
A bioreactor which provides a bioproduct in a near useable form would also be very desirable.
Another shortcoming of current technology is that there are no devices to test various drugs, agents, and compounds effectively against living barrier tissues or showing trans-membrane dispersion of the drug, compound, or agent. An in vitro system for culturing a membrane bearing living tissue would be very desirable. A device to permit the testing of drugs against a living tissue barrier membrane would be very desirable. A device which permits the living tissue membrane to be easily replaced would be very desirable.
It would also be extremely desirable to manufacture a graftable human tissue, such as skin, in vitro. However, previous attempts have not been successful to meet this end. The provision of a graftable human tissue would be extremely desirable.
Rotating Vessels have been used in experimental programs to generate mono-cellular xe2x80x9corganoidsxe2x80x9d. In most experiments the xe2x80x9cmatrixxe2x80x9d supporting the creation of three-dimensional architecture has been beads consisting of man made materials. The success always depended on the cells attaching efficiently to the beads and then being mitotically active enough to initiate aggregation of cell-covered beads. In successful experiments, the cells were generally immortal tumor cell lines and the objective was to create an in vitro model for studying efficacy of chemotherapeutic agentsxe2x80x94anti-cancer drugs. For a number of years therefore very rarely were normal human cells cultured in the rotating vessels and never has a construction and maintenance of poly-cellular tissue such as might be useful for grafting been attempted. A living membrane of normal cells would be very desirable, especially one in the form of a cartridge which could be moved between bioreactors.
Further, although the tumoroids generated in vitro in rotating vessels were useful models to study anti-cancer therapies, since they mimicked an in vivo model better than the cell monolayers did, these organoids were more resistant to anti-cancer drugs or agents. It was also found that the cores in larger tumoroids were necrotic, suggesting that the large size xe2x80x9ctissuesxe2x80x9d may not be receiving their required oxygen and nutrient concentrations. A device which can be used to grow usably sized living tissue would be very desirable.
A cell culture vessel comprising a front vessel half, a back vessel half, an inner tubular member, and a disk-shaped baffle is provided in one embodiment of the invention.
The front vessel half defines a front chamber. The back vessel half defines a back chamber which is generally cylindrical and has a longitudinal axis. The front vessel half is engageable with the back vessel half and comes together with the back vessel half so as to form an annular recess for receipt of a cylindrical sample cartridge rim of a sample cartridge which will separate the front chamber from the back chamber. The inner tubular member extends though an end wall of the back vessel half and into the back chamber along the longitudinal axis. The inner tubular member has an inner end which is positioned in the back chamber. The disk shaped baffle is mounted on the inner end of the tubular member and extends transversely across the back chamber so as to substantially form a partition across the back chamber. The baffle divides the back chamber into a front portion and a back portion. The inner tubular member forms a means for introducing fluid into the front portion of the back chamber. A means for withdrawing a fluid from the back portion of the back chamber is also provided. The invention can be used by positioning a layer of human cells or tissue on a permeable support and flowing culture medium across a cell surface defined by the layer of cells to provide the necessary nourishment.
In another embodiment of the invention, there is provided a sample cartridge which contains a connective tissue equivalent. The sample cartridge comprises a generally annularly shaped washer constructed of a polymeric material and having a first end face and a second end face, a semi-permeable membrane affixed to the first end face of the washer to form an end closure for a cell chamber defined inside of the washer, and a substrate of solidified collagen/fibroblasts mix layered on the semi-permeable membrane in the cell chamber to form the connective tissue equivalent. The sample cartridge can be used to grow epidermal or other types of cells, and can be used for cancer research as well as other types of research. A connective tissue equivalent can be formed with the just described sample cartridge by forming a substrate of solidified collagen/fibroblasts mix on the semi-permeable membrane in the cell chamber.
In another embodiment of the invention, there is provided a cell culture vessel which is useful for the generation of recombinant proteins, antibodies and other bioproducts, and in research. The vessel is formed from a front vessel half, a back vessel half, and a sample cartridge which separates the vessel halves to form two chambers. A growth of secretor cells is maintained in one chamber, while a means for collecting secretions, containing for example proteins, antibodies and other bio-products from the secretor cells, is maintained in a separated vessel chamber designed for collecting the secretions.
The front vessel half defines a front chamber. The back vessel half defines a back chamber which is generally cylindrical and has a longitudinal axis. The front vessel half is sealingly engageable with the back vessel half and comes together with the back vessel half so as to form an annular recess for receipt of a cylindrical sample cartridge rim. The sample cartridge rim is defined by a periphery of a sample cartridge which separates the front chamber from the back chamber. The sample cartridge is formed from a generally annularly shaped washer having a generally cylindrical cartridge rim, a first end face and a second end face. A semi-permeable membrane is affixed to the first end face of the washer and forms an end closure for a cell chamber defined inside of the washer. The sample cartridge is positioned in the cell culture vessel with the rim of the at least one generally cylindrical sample cartridge being positioned in the annular recess so as to separate the front chamber from the back chamber. The growth of secretor cells is maintained in one chamber while a means for collecting secretions from the secretor cells is maintained in the second chamber. The apparatus is preferably utilized by growing the secretor cells and collecting the secretions in adjoining chambers separated by a semi-permeable membrane cartridge.